Poxviruses unlike other DNA viruses replicate in the cytoplasm of the cell and encode many of the enzymes and factors needed for transcription of their genomes. Vaccinia virus, therefore, provides a unique system for combining biochemical and genetic approaches for investigating mechanisms of gene regulation and mRNA biosynthesis. Studies with vaccinia virus indicated that the genes are divided into three temporal classes - early, intermediate and late - that are regulated in a cascade fashion. Progress was made in determining the mechanism of formation of the transcription initiation complex. A functional pre-initiation transcription complex was formed by incubating vaccinia virus early transcription factor VETF and RAP94+ RNA polymerase with an early promoter template immobilized on paramagnetic particles. A preferred order of assembly, VETF followed by RNA polymerase, was demonstrated by stepwise addition experiments. ATP was unnecessary for pre-initiation transcription complex formation but divalent cations were required specifically for the association of RNA polymerase. The ability to form relatively stable pre-initiation complexes in a step-wise manner should allow further characterization of the protein/DNA and protein/protein interactions involved. The vaccinia virus encoded protein VP39 is a poly(A) polymerase subunit, that stimulates the formation of long poly(A) tails, as well as a cap- specific mRNA (nucleoside-2'-O-)-methyltransferase. We have carried out mutagenesis studies aimed at locating regions of VP39 that are important for these activities. Truncation, charge cluster to Ala scanning, and Cys to Ser substitution mutations of VP39 were made and the proteins were synthesized, purified and analyzed.